Processing biomass

ABSTRACT

Biomass (e.g., plant biomass, animal biomass, and municipal waste biomass) is processed to produce useful products, such as fuels. For example, systems are described that can use feedstock materials, such as cellulosic and/or lignocellulosic materials and/or starchy materials, to produce ethanol and/or butanol, e.g., by fermentation.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/139,473 filed Dec. 19, 2008. The complete disclosure of thisprovisional application is hereby incorporated by reference herein.

BACKGROUND

Various carbohydrates, such as cellulosic and lignocellulosic materials,e.g., in fibrous form, are produced, processed, and used in largequantities in a number of applications. Often such materials are usedonce, and then discarded as waste, or are simply considered to be wastematerials, e.g., sewage, bagasse, sawdust, and stover.

Various cellulosic and lignocellulosic materials, their uses, andapplications have been described, for example, in U.S. Pat. Nos.7,307,108, 7,074,918, 6,448,307, 6,258,876, 6,207,729, 5,973,035 and5,952,105.

SUMMARY

Generally, this invention relates to carbohydrate-containing materials(e.g., biomass materials or biomass-derived materials, such as starchymaterials and/or cellulosic or lignocellulosic materials), methods ofmaking and processing such materials to change their structure and/ortheir recalcitrance level, and products made from the changed materials.For example, many of the methods described herein can provide cellulosicand/or lignocellulosic materials that have an oxygen-rich functionality,a lower molecular weight and/or crystallinity relative to a nativematerial. Many of the methods, such as Fenton oxidation methods, providematerials that can be more readily utilized by a variety ofmicroorganisms (with or without enzymatic hydrolysis) to produce usefulproducts, such as hydrogen, alcohols (e.g., ethanol or butanol), organicacids (e.g., acetic acid), hydrocarbons, co-products (e.g., proteins) ormixtures of any of these. Many of the products obtained, such as ethanolor n-butanol, can be utilized as fuel, e.g., as an internal combustionfuel or as a fuel cell feedstock. In addition, the products describedherein can be utilized for electrical power generation, e.g., in aconventional steam generating plant or in a fuel cell plant.

In one aspect, the invention features methods of changing molecularstructures and/or reducing recalcitrance in materials, such ashydrocarbon-containing materials and/or biomass materials, e.g.,cellulosic or lignocellulosic materials, such as any one or moreunprocessed (e.g., cut grass), semi-processed (e.g., comminuted grass)or processed materials (e.g., comminuted and irradiated grass) describedherein.

The methods can feature oxidative methods of reducing recalcitrance incellulosic or lignocellulosic materials that employ Fenton-typechemistry. Fenton-type chemistry is discussed in Pestovsky et al.,Angew. Chem., Int. Ed. 2005, 44, 6871-6874, the entire disclosure ofwhich is hereby incorporated by reference herein. The methods can alsofeature combinations of Fenton oxidation and any other pretreatmentmethod described herein in any order.

Without wishing to be bound by any particular theory, it is believedthat oxidation increases the number of hydrogen-bonding groups on thecellulose and/or the lignin, such as hydroxyl groups, aldehyde groups,ketone groups carboxylic acid groups or anhydride groups, which canincrease its dispersability and/or its solubility.

In one aspect, the invention features methods that include contacting,in a mixture, a first cellulosic or lignocellulosic material having afirst level of recalcitrance with one or more compounds comprising oneor more naturally-occurring, non-radioactive metallic elements, e.g.,non-radioactive group 5, 6, 7, 8, 9, 10 or 11 elements, and, optionally,one or more oxidants capable of increasing an oxidation state of atleast some of said elements, to produce a second cellulosic orlignocellulosic material having a second level of recalcitrance lowerthan the first level of recalcitrance.

Other methods include combining a hydrocarbon-containing material withone or more compounds including one or more naturally-occurring,non-radioactive metallic elements, e.g., non-radioactive group 5, 6, 7,8, 9, 10 or 11 elements to provide a mixture in which the one or morecompounds contact the hydrocarbon-containing material; and maintainingthe contact for a period of time and under conditions sufficient tochange the structure of the hydrocarbon-containing material.

In some embodiments, the method further includes combining the firstcellulosic, lignocellulosic, or hydrocarbon-containing material with oneor more oxidants capable of increasing an oxidation state of at leastsome of the elements. In such instances, the one or more oxidantscontact the material with the one or more compounds in the mixture. Insome embodiments, the one or more oxidants include ozone and/or hydrogenperoxide.

In some embodiments, the one or more elements are in a 1+, 2+, 3+, 4+ or5+ oxidation state. In particular instances, the one or more elementsare in a 2+, 3+ or 4+ oxidation state. For example, iron can be in theform of iron(II), iron(III) or iron(IV).

In particular instances, the one or more elements include Mn, Fe, Co,Ni, Cu or Zn, preferably Fe or Co. For example, the Fe or Co can be inthe form of a sulfate, e.g., iron(II) or iron(III) sulfate.

In some embodiments, the one or more oxidants are applied to the firstcellulosic or lignocellulosic material and the one or more compounds asa gas, such as by generating ozone in-situ by irradiating the firstcellulosic or lignocellulosic material and the one or more compoundsthrough air with a beam of particles, such as electrons or protons.

In some embodiments, the mixture further includes one or morehydroquinones, such as 2,5-dimethoxyhydroquinone and/or one or morebenzoquinones, such as 2,5-dimethoxy-1,4-benzoquinone. Such compounds,which have similar molecular entities as lignin, can aid in electrontransfer.

In some desirable embodiments, the one or more oxidants areelectrochemically or electromagnetically generated in-situ. For example,hydrogen peroxide and/or ozone can be electrochemically orelectromagnetically produced within a contact or reaction vessel oroutside the vessel and transferred into the vessel.

The methods may further include contacting the second cellulosic orlignocellulosic material with an enzyme and/or microorganism. Productsproduced by such contact can include any of those products describedherein, such as food or fuel, e.g., ethanol, or any other productsdescribed in U.S. Provisional Application Ser. No. 61/139,453, which ishereby incorporated by reference herein in its entirety.

In another aspect, the invention features systems that include astructure or carrier, e.g., a reaction vessel, containing a mixtureincluding 1) any material described herein, such as a cellulosic orlignocellulosic material and 2) one or more compounds comprising one ormore naturally-occurring, non-radioactive metallic elements, e.g.,non-radioactive group 5, 6, 7, 8, 9, 10 or 11 elements. Optionally, themixture can include 3) one or more oxidants capable of increasing anoxidation state of at least some of the elements.

In another aspect, the invention features compositions that include 1)any material described herein, such as a cellulosic or lignocellulosicmaterial and 2) one or more compounds comprising one or morenaturally-occurring, non-radioactive group 5, 6, 7, 8, 9, 10 or 11elements. Optionally, the composition can include one or more oxidantscapable of increasing an oxidation state of at least some of theelements.

In another aspect, the invention features methods of changing molecularstructures and/or reducing recalcitrance in biomass materials, such ascellulosic or lignocellulosic materials. The methods include combining afirst lignocellulosic material having a first level of recalcitrancewith one or more ligninases and/or one or more biomass-destroying, e.g.,lignin-destroying organisms, in a manner that the one or more ligninasesand/or organisms contact the first cellulosic or lignocellulosicmaterial; and maintaining the contact for a period of time and underconditions sufficient to produce a second lignocellulosic materialhaving a second level of recalcitrance lower than the first level ofrecalcitrance. The method can further include contacting the secondcellulosic or lignocellulosic material with an enzyme and/ormicroorganism, e.g., to make any product described herein, e.g., food orfuel, e.g., ethanol or butanol (e.g., n-butanol) or any productdescribed in U.S. Provisional Application Ser. No. 61/139,453.

The ligninase can be, e.g., one or more of manganese peroxidase, ligninperoxidase or laccases.

The biomass-destroying organism can be, e.g., one or more of white rot,brown rot or soft rot. For example, the biomass-destroying organism canbe a Basidiomycetes fungus. In particular embodiments, thebiomass-destroying organism is Phanerochaete chrysoporium or Gleophyllumtrabeum.

In certain embodiments, the first material is in the form of a fibrousmaterial that includes fibers provided by shearing a fiber source.Shearing alone can reduce the crystallinity of a fibrous material andcan work synergistically with any process technique that also reducescrystallinity and/or molecular weight. For example, the shearing can beperformed with a rotary knife cutter. In some embodiments, the fibrousmaterial has an average length-to-diameter ratio of greater than 5/1.

The first and/or second material can have, e.g., a BET surface area ofgreater than 0.25 m²/g and/or a porosity of greater than about 25percent.

To further aid in the reduction of the molecular weight of thecellulose, an enzyme, e.g., a cellulolytic enzyme, or a chemical, e.g.,sodium hypochlorite, an acid, a base or a swelling agent, can beutilized with any method described herein.

When a microorganism is utilized, it can be a natural microorganism oran engineered microorganism. For example, the microorganism can be abacterium, e.g., a cellulolytic bacterium, a fungus, e.g., a yeast, anenzyme, a plant or a protist, e.g., an algae, a protozoa or afungus-like protist, e.g., a slime mold. When the organisms arecompatible, mixtures may be utilized. Generally, various microorganismscan produce a number of useful products, such as a fuel, by operatingon, e.g., fermenting the materials. For example, alcohols, organicacids, hydrocarbons, hydrogen, proteins or mixtures of any of thesematerials can be produced by fermentation or other processes.

Examples of products that may be produced include mono- andpolyfunctional C1-C6 alkyl alcohols, mono- and poly-functionalcarboxylic acids, C1-C6 hydrocarbons, and combinations thereof. Specificexamples of suitable alcohols include methanol, ethanol, propanol,isopropanol, butanol, ethylene glycol, propylene glycol, 1,4-butanediol, glycerin, and combinations thereof. Specific example of suitablecarboxylic acids include formic acid, acetic acid, propionic acid,butyric acid, valeric acid, caproic acid, palmitic acid, stearic acid,oxalic acid, malonic acid, succinic acid, glutaric acid, oleic acid,linoleic acid, glycolic acid, lactic acid, γ-hydroxybutyric acid, andcombinations thereof. Examples of suitable hydrocarbons include methane,ethane, propane, pentane, n-hexane, and combinations thereof. Many ofthese products may be used as fuels.

The term “fibrous material,” as used herein, is a material that includesnumerous loose, discrete and separable fibers. For example, a fibrousmaterial can be prepared from a bleached Kraft paper fiber source byshearing, e.g., with a rotary knife cutter.

The term “screen,” as used herein, means a member capable of sievingmaterial according to size. Examples of screens include a perforatedplate, cylinder or the like, or a wire mesh or cloth fabric.

The term “pyrolysis,” as used herein, means to break bonds in a materialby the application of heat energy. Pyrolysis can occur while the subjectmaterial is under vacuum, or immersed in a gaseous material, such as anoxidizing gas, e.g., air or oxygen, or a reducing gas, such as hydrogen.

Oxygen content is measured by elemental analysis by pyrolyzing a samplein a furnace operating at 1300° C. or above.

Examples of biomass feedstock include paper, paper products, paperwaste, wood, wood wastes and residues, particle board, sawdust,agricultural waste and crop residues, sewage, silage, grasses, ricehulls, bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw,corn cobs, corn stover, switchgrass, alfalfa, hay, rice hulls, coconuthair, cotton, synthetic celluloses, seaweed, algae, municipal waste, ormixtures of these. The biomass can be or can include a natural or asynthetic material.

The terms “plant biomass” and “lignocellulosic biomass” refer tovirtually any plant-derived organic matter (woody or non-woody).

For the purposes of this disclosure, carbohydrates are materials thatare composed entirely of one or more saccharide units or that includeone or more saccharide units. Carbohydrates can be polymeric (e.g.,equal to or greater than 10-mer, 100-mer, 1,000-mer, 10,000-mer, or100,000-mer), oligomeric (e.g., equal to or greater than a 4-mer, 5-mer,6-mer, 7-mer, 8-mer, 9-mer or 10-mer), trimeric, dimeric, or monomeric.When the carbohydrates are formed of more than a single repeat unit,each repeat unit can be the same or different. Examples of polymericcarbohydrates include cellulose, xylan, pectin, and starch, whilecellobiose and lactose are examples of dimeric carbohydrates. Examplesof monomeric carbohydrates include glucose and xylose. Carbohydrates canbe part of a supramolecular structure, e.g., covalently bonded into thestructure. Examples of such materials include lignocellulosic materials,such as that found in wood.

A starchy material is one that is or includes significant amounts ofstarch or a starch derivative, such as greater than about 5 percent byweight starch or starch derivative. For purposes of this disclosure, astarch is a material that is or includes an amylose, an amylopectin, ora physical and/or chemical mixture thereof, e.g., a 20:80 or 30:70percent by weight mixture of amylose to amylopectin. For example, rice,corn, and mixtures thereof are starchy materials. Starch derivativesinclude, e.g., maltodextrin, acid-modified starch, base-modified starch,bleached starch, oxidized starch, acetylated starch, acetylated andoxidized starch, phosphate-modified starch, genetically-modified starchand starch that is resistant to digestion.

Swelling agents as used herein are materials that cause a discernableswelling, e.g., a 2.5 percent increase in volume over an unswollen stateof cellulosic and/or lignocellulosic materials, when applied to suchmaterials as a solution, e.g., a water solution. Examples includealkaline substances, such as sodium hydroxide, potassium hydroxide,lithium hydroxide and ammonium hydroxides, acidifying agents, such asmineral acids (e.g., sulfuric acid, hydrochloric acid and phosphoricacid), salts, such as zinc chloride, calcium carbonate, sodiumcarbonate, benzyltrimethylammonium sulfate, and basic organic amines,such as ethylene diamine.

A “sheared material,” as used herein, is a material that includesdiscrete fibers in which at least about 50% of the discrete fibers, havea length/diameter (L/D) ratio of at least about 5, and that has anuncompressed bulk density of less than about 0.6 g/cm³. A shearedmaterial is thus different from a material that has been cut, chopped orground.

Changing a molecular structure of a biomass feedstock, as used herein,means to change the chemical bonding arrangement or conformation of thestructure. For example, the change in the molecular structure caninclude changing the supramolecular structure of the material, oxidationof the material, changing an average molecular weight, changing anaverage crystallinity, changing a surface area, changing a degree ofpolymerization, changing a porosity, changing a degree of branching,grafting on other materials, changing a crystalline domain size, or anchanging an overall domain size.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein or attachedhereto are incorporated by reference in their entirety for all that theycontain.

Any biomass material, e.g., carbohydrate-containing material, e.g.,cellulosic and/or lignocellulosic material described herein can beutilized in any application or process described in any patent or patentapplication incorporated by reference herein.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is block diagram illustrating conversion of a fiber source into afirst and second fibrous material.

FIG. 2 is a cross-sectional view of a rotary knife cutter.

FIG. 3 is block diagram illustrating conversion of a fiber source into afirst, second and third fibrous material.

FIG. 4 is a schematic cross-sectional side view of a reactor.

FIG. 5 shows a sequence of chemical reactions illustrating Fentonchemistry.

FIG. 6 shows a sequence of Fenton reactions illustrating conversion ofbenzene to phenol and toluene to benzaldehyde and benzyl alcohol.

FIG. 7 shows a reaction scheme for the preparation of a reactive iron(IV) compound from an iron (II) compound.

FIG. 8 shows a proposed pathway for reduction of Fe (III) and productionof hydrogen peroxide in the presence of 2,5-dimethoxyhydroquinone.

FIG. 9 is a scanning electron micrograph of a fibrous material producedfrom polycoated paper at 25× magnification. The fibrous material wasproduced on a rotary knife cutter utilizing a screen with ⅛ inchopenings.

FIG. 10 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was produced on a rotary knife cutter utilizing a screen with ⅛inch openings.

FIG. 11 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was twice sheared on a rotary knife cutter utilizing a screenwith 1/16 inch openings during each shearing.

FIG. 12 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was thrice sheared on a rotary knife cutter. During the firstshearing, a ⅛ inch screen was used; during the second shearing, a 1/16inch screen was used, and during the third shearing a 1/32 inch screenwas used.

DETAILED DESCRIPTION

Using the methods described herein, biomass can be processed to a lowerlevel of recalcitrance and converted into useful products such as fuels.Systems and processes are described below that can use as feedstocksmaterials such as cellulosic and/or lignocellulosic materials that arereadily available, but can be difficult to process, for example, bysaccharification and/or by fermentation. In some implementations thefeedstock materials are first physically prepared for processing, forexample by size reduction. The physically prepared feedstock is thenpretreated using oxidation (e.g., using Fenton-type chemistry), and mayin some cases be further treated with one or more of radiation,sonication, pyrolysis, and steam explosion. Alternatively, in somecases, the feedstock is first treated with one or more of radiation,sonication, pyrolysis, and steam explosion, and then treated usingoxidation, e.g., Fenton-type chemistry.

Preferred oxidative methods for reducing recalcitrance in cellulosic orlignocellulosic materials include Fenton-type chemistry, discussedabove, in which one or more group 5, 6, 7, 8, 9, 10 or 11 elements,optionally along with one or more oxidants capable of increasing anoxidation state of at least some of the elements are utilized.

After pretreatment, the pretreated material can be further processed,e.g., using primary processes such as saccharification and/orfermentation, to produce a product.

Types of Biomass

Generally, any biomass material that is or includes carbohydratescomposed entirely of one or more saccharide units or that include one ormore saccharide units can be processed by any of the methods describedherein. For example, the biomass material can be cellulosic orlignocellulosic materials, or starchy materials, such as kernels ofcorn, grains of rice or other foods.

Fiber sources include cellulosic fiber sources, including paper andpaper products (e.g., polycoated paper and Kraft paper), andlignocellulosic fiber sources, including wood, and wood-relatedmaterials, e.g., particle board. Other suitable fiber sources includenatural fiber sources, e.g., grasses, rice hulls, bagasse, cotton, jute,hemp, flax, bamboo, sisal, abaca, straw, corn cobs, rice hulls, coconuthair; fiber sources high in α-cellulose content, e.g., cotton; andsynthetic fiber sources, e.g., extruded yarn (oriented yarn orun-oriented yarn). Natural or synthetic fiber sources can be obtainedfrom virgin scrap textile materials, e.g., remnants or they can be postconsumer waste, e.g., rags. When paper products are used as fibersources, they can be virgin materials, e.g., scrap virgin materials, orthey can be post-consumer waste. Aside from virgin raw materials,post-consumer, industrial (e.g., offal), and processing waste (e.g.,effluent from paper processing) can also be used as fiber sources. Also,the fiber source can be obtained or derived from human (e.g., sewage),animal or plant wastes. Additional fiber sources have been described inU.S. Pat. Nos. 6,448,307, 6,258,876, 6,207,729, 5,973,035 and 5,952,105,the full disclosures of which are incorporated by reference herein.

Starchy materials include starch itself, e.g., corn starch, wheatstarch, potato starch or rice starch, a derivative of starch, or amaterial that includes starch, such as an edible food product or a crop.For example, the starchy material can be arracacha, buckwheat, banana,barley, cassava, kudzu, oca, sago, sorghum, regular household potatoes,sweet potato, taro, yams, or one or more beans, such as favas, lentilsor peas. Blends of any one or more starchy material is also a starchymaterial. In particular embodiments, the starchy material is derivedfrom corn. Various corn starches and derivatives are described in “CornStarch,” Corn Refiners Association (11^(th) Edition, 2006), which ishereby incorporated by reference herein.

Blends of any biomass materials described herein can be utilized formaking any of the products described herein, such as ethanol. Forexample, blends of cellulosic materials and starchy materials can beutilized for making any product described herein.

Feed Preparation

In some cases, methods of processing begin with a physical preparationof the feedstock, e.g., size reduction of raw feedstock materials, suchas by cutting, grinding, shearing or chopping. In some cases, loosefeedstock (e.g., recycled paper, starchy materials, or switchgrass) isprepared by shearing or shredding. Screens and/or magnets can be used toremove oversized or undesirable objects such as, for example, rocks ornails from the feed stream.

Feed preparation systems can be configured to produce feed streams withspecific characteristics such as, for example, specific maximum sizes,specific length-to-width, or specific surface areas ratios. As a part offeed preparation, the bulk density of feedstocks can be controlled(e.g., increased). If desired, lignin can be removed from any feedstockthat includes lignin.

Size Reduction

In some embodiments, the material to be processed is in the form of afibrous material that includes fibers provided by shearing a fibersource. For example, the shearing can be performed with a rotary knifecutter.

For example, and by reference to FIG. 1, a fiber source 210 is sheared,e.g., in a rotary knife cutter, to provide a first fibrous material 212.The first fibrous material 212 is passed through a first screen 214having an average opening size of 1.59 mm or less ( 1/16 inch, 0.0625inch) to provide a second fibrous material 216. If desired, fiber sourcecan be cut prior to the shearing, e.g., with a shredder.

In some embodiments, the shearing of fiber source and the passing of theresulting first fibrous material through first screen are performedconcurrently. The shearing and the passing can also be performed in abatch-type process.

For example, a rotary knife cutter can be used to concurrently shear thefiber source and screen the first fibrous material. Other methods ofmaking the fibrous materials include, e.g., stone grinding, mechanicalripping or tearing, pin grinding or air attrition milling. Referring toFIG. 2, a rotary knife cutter 220 includes a hopper 222 that can beloaded with a shredded fiber source 224. The shredded fiber source issheared between stationary blades 230 and rotating blades 232 to providea first fibrous material 240. First fibrous material 240 passes throughscreen 242, and the resulting second fibrous material 244 is captured inbin 250. To aid in the collection of the second fibrous material, avacuum source 252 can be utilized to maintain the bin at a pressurebelow nominal atmospheric pressure, e.g., at least 10, 25, 50 or 75percent below nominal atmospheric pressure.

Shearing can be advantageous for “opening up” and “stressing” thefibrous materials, making the cellulose of the materials moresusceptible to chain scission and/or reduction of crystallinity. Theopen materials can also be more susceptible to oxidation.

The fiber source can be sheared in a dry state, a hydrated state (e.g.,having up to ten percent by weight absorbed water), or in a wet state,e.g., having between about 10 percent and about 75 percent by weightwater. The fiber source can even be sheared while partially or fullysubmerged under a liquid, such as water, ethanol, isopropanol. The fibersource can also be sheared under a gas (such as a stream or atmosphereof gas other than air), e.g., oxygen or nitrogen, or steam.

In some embodiments, the average opening size of the first screen 214 isless than 0.79 mm (0.031 inch), e.g., less than 0.51 mm (0.020 inch),0.40 mm (0.015 inch), 0.23 mm (0.009 inch), 0.20 mm (0.008 inch), 0.18mm (0.007 inch), 0.13 mm (0.005 inch), or even less than less than 0.10mm (0.004 inch). The characteristics of suitable screens are described,for example, in US 2008-0206541. In some embodiments, the open area ofthe mesh is less than 52%, e.g., less than 41%, less than 36%, less than31%, or less than 30%.

In some embodiments, the second fibrous is sheared and passed throughthe first screen, or a different sized screen. In some embodiments, thesecond fibrous material is passed through a second screen having anaverage opening size equal to or less than that of first screen.Referring to FIG. 3, a third fibrous material 220 can be prepared fromthe second fibrous material 216 by shearing the second fibrous material216 and passing the resulting material through a second screen 222having an average opening size less than the first screen 214. In suchinstances, a ratio of the average length-to-diameter ratio of the secondfibrous material to the average length-to-diameter ratio of the thirdfibrous material can be, e.g., less than 1.5, e.g., less than 1.4, lessthan 1.25, or even less than 1.1.

Generally, the fibers of the fibrous materials can have a relativelylarge average length-to-diameter ratio (e.g., greater than 20-to-1),even if they have been sheared more than once. In addition, the fibersof the fibrous materials described herein may have a relatively narrowlength and/or length-to-diameter ratio distribution.

As used herein, average fiber widths (i.e., diameters) are thosedetermined optically by randomly selecting approximately 5,000 fibers.Average fiber lengths are corrected length-weighted lengths. BET(Brunauer, Emmet and Teller) surface areas are multi-point surfaceareas, and porosities are those determined by mercury porosimetry.

The average length-to-diameter ratio of the second fibrous material 14can be, e.g. greater than 8/1, e.g., greater than 10/1, greater than15/1, greater than 20/1, greater than 25/1, or greater than 50/1. Anaverage length of the second fibrous material 14 can be, e.g., betweenabout 0.5 mm and 2.5 mm, e.g., between about 0.75 mm and 1.0 mm, and anaverage width (i.e., diameter) of the second fibrous material 14 can be,e.g., between about 5 μm and 50 μm, e.g., between about 10 μm and 30 μm.

In some embodiments, a standard deviation of the length of the secondfibrous material 14 is less than 60 percent of an average length of thesecond fibrous material 14, e.g., less than 50 percent of the averagelength, less than 40 percent of the average length, less than 25 percentof the average length, less than 10 percent of the average length, lessthan 5 percent of the average length, or even less than 1 percent of theaverage length.

In some embodiments, a BET surface area of the second fibrous materialis greater than 0.1 m²/g, e.g., greater than 0.25 m²/g, greater than 0.5m²/g, greater than 1.0 m²/g, greater than 1.5 m²/g, greater than 1.75m²/g, greater than 5.0 m²/g, greater than 10 m²/g, greater than 25 m²/g,greater than 35 m²/g, greater than 50 m²/g, greater than 60 m²/g,greater than 75 m²/g, greater than 100 m²/g, greater than 150 m²/g,greater than 200 m²/g, or even greater than 250 m²/g.

A porosity of the second fibrous material 14 can be, e.g., greater than20, 25, 35, 50, 60, 70, 80, 85, 90, 92, 94, 95, 97.5 or 99 percent, oreven greater than 99.5 percent.

In some embodiments, a ratio of the average length-to-diameter ratio ofthe first fibrous material to the average length-to-diameter ratio ofthe second fibrous material is, e.g., less than 1.5, e.g., less than1.4, less than 1.25, less than 1.1, less than 1.075, less than 1.05,less than 1.025, or even substantially equal to 1.

In some embodiments, the third fibrous material is passed through athird screen to produce a fourth fibrous material. The fourth fibrousmaterial can be, e.g., passed through a fourth screen to produce a fifthmaterial. Similar screening processes can be repeated as many times asdesired to produce the desired fibrous material having the desiredproperties.

In some implementations, the size reduction equipment may be portable,e.g., in the manner of the mobile processing equipment described in U.S.Provisional Patent Application Ser. 60/832,735, now PublishedInternational Application No. WO 2008/011598.

Pretreatment

Physically prepared feedstock can be pretreated for use in primaryproduction processes such as saccharification and fermentation by, forexample, reducing the average molecular weight and crystallinity of thefeedstock and/or increasing the surface area and/or porosity of thefeedstock. Pretreatment processes include utilizing Fenton-typechemistry, discussed above, and can further include one or more ofirradiation, sonication, oxidation, pyrolysis, and steam explosion.

Fenton Chemistry

In some embodiments, the one or more elements used in the Fentonreaction are in a 1+, 2+, 3+, 4+ or 5+ oxidation state. In particularinstances, the one or more elements include Mn, Fe, Co, Ni, Cu or Zn,preferably Fe or Co. For example, the Fe or Co can be in the form of asulfate, e.g., iron(II) or iron(III) sulfate. In particular instances,the one or more elements are in a 2+, 3+ or 4+ oxidation state. Forexample, iron can be in the form of iron(II), iron(III) or iron(IV).

Exemplary iron (II) compounds include ferrous sulfate heptahydrate,iron(II) acetylacetonate, (+)-iron(II) L-ascorbate, iron(II) bromide,iron(II) chloride, iron(II) chloride hydrate, iron(II) chloridetetrahydrate, iron(II) ethylenediammonium sulfate tetrahydrate, iron(II)fluoride, iron(II) gluconate hydrate, iron(II) D-gluconate dehydrate,iron(II) iodide, iron(II) lactate hydrate, iron(II) molybdate, iron(II)oxalate dehydrate, iron(II) oxide, iron(II,III) oxide, iron(II)perchlorate hydrate, iron(II) phthalocyanine, iron(II) phthalocyaninebis(pyridine) complex, iron(II) sulfate heptahydrate, iron(II) sulfatehydrate, iron(II) sulfide, iron(II) tetrafluoroborate hexahydrate,iron(II) titanate, ammonium iron(II) sulfate hexahydrate, ammoniumiron(II) sulfate, cyclopentadienyl iron(II) dicarbonyl dimer,ethylenediaminetetraacetic acid hydrate iron(III) sodium salt and ferriccitrate.

Exemplary iron (III) compounds include iron(III) acetylacetonate,iron(III) bromide, iron(III) chloride, iron(III) chloride hexahydrate,iron(III) chloride solution, iron(III) chloride on silica gel, iron(III)citrate, tribasic monohydrate, iron(III) ferrocyanide, iron(III)fluoride, iron(III) fluoride trihydrate, iron(III) nitrate nonahydrate,iron(III) nitrate on silica gel, iron(III) oxalate hexahydrate,iron(III) oxide, iron(III) perchlorate hydrate, iron(III) phosphate,iron(III) phosphate dehydrate, iron(III) phosphate hydrate, iron(III)phosphate tetrahydrate, iron(III) phthalocyanine chloride, iron(III)phthalocyanine-4,4′,4″, 4′″-tetrasulfonic acid, compound with oxygenhydrate monosodium salt, iron(III) pyrophosphate, iron(III) sulfatehydrate, iron(III) p-toluenesulfonate hexahydrate, iron(III)tris(2,2,6,6-tetramethyl-3,5-heptanedionate) and ammonium iron(III)citrate.

Exemplary cobalt (II) compounds include cobalt(II) acetate, cobalt(II)acetate tetrahydrate, cobalt(II) acetylacetonate hydrate, cobalt(II)benzoylacetonate, cobalt(II) bromide, cobalt(II) bromide hydrate andcobalt(II) carbonate hydrate.

Exemplary cobalt (III) compounds include cobalt(III) acetylacetonate,cobalt(III) fluoride, cobalt(III) oxide, cobalt(III) sepulchratetrichloride, hexamine cobalt(III) chloride,bis(cyclopentadienyl)cobalt(III) hexafluorophosphate andbis(ethylcyclopentadienyl)cobalt(III) hexafluorophosphate.

Exemplary oxidants include peroxides, such as hydrogen peroxide andbenzoyl peroxide, persulfates, such as ammonium persulfate, activatedforms of oxygen, such as ozone, permanganates, such as potassiumpermanganate, perchlorates, such as sodium perchlorate, andhypochlorites, such as sodium hypochlorite (household bleach).

Generally, Fenton oxidation occurs in an oxidizing environment. Forexample, the oxidation can be effected or aided by pyrolysis in anoxidizing environment, such as in air or argon enriched in air. To aidin the oxidation, various chemical agents, such as oxidants, acids orbases can be added to the material prior to or during oxidation. Forexample, a peroxide (e.g., benzoyl peroxide) can be added prior tooxidation.

In some cases, pH is maintained at or below about 5.5 during contact,such as between 1 and 5, between 2 and 5, between 2.5 and 5 or betweenabout 3 and 5. The contact period may be, for example, between 2 and 12hours, e.g., between 4 and 10 hours or between 5 and 8 hours. In someinstances, the reaction conditions are controlled so that thetemperature does not exceed 300° C., e.g., the temperature remains lessthan 250, 200, 150, 100 or even less than 50° C. In some cases, thetemperature remains substantially ambient, e.g., at or about 20-25° C.

Referring to FIG. 4, reactive mixtures 2108 within a vessel 2110 can beprepared using various approaches. For example, in instances in whichthe mixture includes one or more compounds and one or more oxidants, thefirst cellulosic or lignocellulosic material can be first dispersed inwater or an aqueous medium, and then the one or more compounds can beadded, followed by addition of the one or more oxidants. Alternatively,the one or more oxidants can added, followed by the one or morecompounds, or the one or more oxidants and the one or more compounds canbe concurrently added separately to the dispersion (e.g., each addedindependently through a separate addition device 2120, 2122 to thedispersion).

In some embodiments, a total maximum concentration of the elements inthe one or more compounds measured in the dispersion is from about 10 μMto about 500 mM, e.g., between about 25 μM and about 250 mM or betweenabout 100 μM and about 100 mM, and/or a total maximum concentration ofthe one or more oxidants is from about 100 μM to about 1 M, e.g.,between about 250 μM and about 500 mM, or between about 500 μm and 250mM. In some embodiments, the mole ratio of the elements in the one ormore compounds to the one or more oxidants is from about 1:1000 to about1:25, such as from about 1:500 to about 1:25 or from about 1:100 toabout 1:25.

In some cases, the one or more oxidants are applied to the firstcellulosic or lignocellulosic material and the one or more compounds asa gas, such as by generating ozone in-situ by irradiating the firstcellulosic or lignocellulosic and the one or more compounds through airwith a beam of particles, such as electrons or protons.

In other cases, the first cellulosic or lignocellulosic material isfirst dispersed in water or an aqueous medium that includes the one ormore compounds dispersed and/or dissolved therein, and then water isremoved after a soak time (e.g., loose and free water is removed byfiltration), and then the one or more oxidants are applied to thecombination as a gas, such as by generating ozone in-situ by irradiatingthe first cellulosic or lignocellulosic and the one or more compoundsthrough air with a beam of particles, such as electrons (e.g., eachbeing accelerated by a potential difference of between 3 MeV and 10MeV).

Referring now to FIG. 5, in some particular embodiments, an iron (II)compound is utilized for the Fenton-type chemistry, such as iron (II)sulfate, and hydrogen peroxide is utilized as the oxidant. FIG. 5illustrates that in such a system, hydrogen peroxide oxidizes the iron(II) to generate iron (III), hydroxyl radicals and hydroxide ions(equation 1). The hydroxyl radicals can then react with the firstcellulosic or lignocellulosic material, thereby oxidizing it to thesecond cellulosic or lignocellulosic material. The iron (III) thusproduced can be reduced back to iron (II) by the action of hydrogenperoxide and hydroperoxyl radicals (equations 2 and 3). Equation 4illustrates that it is also possible for an organic radical (R) toreduce iron (III) back to iron (II).

FIG. 6 illustrates that iron (II) sulfate and hydrogen peroxide inaqueous solutions and at pH below about 6 can oxidize aromatic rings togive phenols, aldehydes and alcohols. When applied to cellulosic orlignocellulosic material, these Fenton-type reactions can help enhancethe solubility of the lignocellulosic material by functionalization ofthe lignin and/or cellulose or hemicellulose, and by reduction inmolecular weight of the lignocellulosic material. The net effect of theFenton-type reactions on the lignocellulosic material can be a change inmolecular structure and/or a reduction in its recalcitrance.

FIG. 7 shows that hydrated iron (II) compounds, such as hydrated iron(II) sulfate, can react with ozone in aqueous solutions to generateextremely reactive hydrated iron (IV) compounds that can react with andoxidize cellulosic and lignocellulosic materials.

In some desirable embodiments, the mixture further includes one or morehydroquinones, such as 2,5-dimethoxyhydroquinone (DMHQ) and/or one ormore benzoquinones, such as 2,5-dimethoxy-1,4-benzoquinone (DMBQ), whichcan aid in electron transfer reactions. FIG. 8 illustrates how iron(III) can be reduced by DMHQ to give iron (II) and DMHQ semi-quinoneradical. Addition of oxygen to the semi-quinone then givesalpha-hydroxyperoxyl radical that eliminates HOO. to give DMBQ. Finally,HOO. oxidizes iron (II) or dismutates to generate hydrogen peroxide.

In some desirable embodiments, the one or more oxidants areelectrochemically or electromagnetically generated in-situ. For example,hydrogen peroxide and/or ozone can be electrochemically orelectromagnetically produced within a contact or reaction vessel.

In some implementations, the Fenton reaction vessel may be portable,e.g., in the manner of the mobile processing equipment described in U.S.Provisional Patent Application Ser. 60/832,735, now PublishedInternational Application No. WO 2008/011598.

Radiation Treatment

Before, during or after the Fenton oxidation discussed above, one ormore irradiation processing sequences can be used to pretreat thefeedstock. Irradiation can reduce the molecular weight and/orcrystallinity of feedstock. In some embodiments, energy deposited in amaterial that releases an electron from its atomic orbital is used toirradiate the materials. The radiation may be provided by 1) heavycharged particles, such as alpha particles or protons, 2) electrons,produced, for example, in beta decay or electron beam accelerators, or3) electromagnetic radiation, for example, gamma rays, x rays, orultraviolet rays. In one approach, radiation produced by radioactivesubstances can be used to irradiate the feedstock. In some embodiments,any combination in any order or concurrently of (1) through (3) may beutilized. In another approach, electromagnetic radiation (e.g., producedusing electron beam emitters) can be used to irradiate the feedstock.The doses applied depend on the desired effect and the particularfeedstock. For example, high doses of radiation can break chemical bondswithin feedstock components and low doses of radiation can increasechemical bonding (e.g., cross-linking) within feedstock components. Insome instances when chain scission is desirable and/or polymer chainfunctionalization is desirable, particles heavier than electrons, suchas protons, helium nuclei, argon ions, silicon ions, neon ions carbonions, phosphorus ions, oxygen ions or nitrogen ions can be utilized.When ring-opening chain scission is desired, positively chargedparticles can be utilized for their Lewis acid properties for enhancedring-opening chain scission. For example, when maximum oxidation isdesired, oxygen ions can be utilized, and when maximum nitration isdesired, nitrogen ions can be utilized.

Doses

In some embodiments, the irradiating (with any radiation source or acombination of sources) is performed until the material receives a doseof at least 0.25 Mrad, e.g., at least 1.0 Mrad, 2.5 Mrad, 5.0 Mrad, 10.0Mrad, 25 Mrad, 50 Mrad, or even at least 100 Mrad. In some embodiments,the irradiating is performed until the material receives a dose ofbetween 1.0 Mrad and 6.0 Mrad, e.g., between 1.5 Mrad and 4.0 Mrad.

In some embodiments, the irradiating is performed at a dose rate ofbetween 5.0 and 1500.0 kilorads/hour, e.g., between 10.0 and 750.0kilorads/hour or between 50.0 and 350.0 kilorads/hours.

In some embodiments, two or more radiation sources are used, such as twoor more ionizing radiations. For example, samples can be treated, in anyorder, with a beam of electrons, followed by gamma radiation and UVlight having wavelengths from about 100 nm to about 280 nm.

In some embodiments, relatively low doses of radiation can crosslink,graft, or otherwise increase the molecular weight of acarbohydrate-containing material, such as a cellulosic orlignocellulosic material (e.g., cellulose). For example, a fibrousmaterial that includes a first cellulosic and/or lignocellulosicmaterial having a first molecular weight can be irradiated in such amanner as to provide a second cellulosic and/or lignocellulosic materialhaving a second molecular weight higher than the first molecular weight.For example, if gamma radiation is utilized as the radiation source, adose of from about 1 Mrad to about 10 Mrad, about 1 Mrad to about 75Mrad, or about 1 Mrad to about 100 Mrad can be applied. In someimplementations, from about 1.5 Mrad to about 7.5 Mrad or from about 2.0Mrad to about 5.0 Mrad, can be applied.

Sonication, Pyrolysis, Oxidation, and Steam Explosion

One or more sonication, pyrolysis, oxidative processing, and/or steamexplosion can be used to further pretreat the feedstock. Such processingcan reduce the molecular weight and/or crystallinity of feedstock andbiomass, e.g., one or more carbohydrate sources, such as cellulosic orlignocellulosic materials, or starchy materials. These processes aredescribed in detail in U.S. Ser. No. 12/429,045.

In some embodiments, biomass can be processed by applying two or more ofany of the processes described herein, such Fenton oxidation combinedwith any one, two or more of radiation, sonication, oxidation,pyrolysis, and steam explosion either with or without prior,intermediate, or subsequent physical feedstock preparation. Theprocesses can be applied in any order or concurrently to the biomass.Multiple processes can in some cases provide materials that can be morereadily utilized by a variety of microorganisms because of their lowermolecular weight, lower crystallinity, and/or enhanced solubility.Multiple processes can provide synergies and can reduce overall energyinput required in comparison to any single process.

Primary Processing

Primary processing of the pretreated feedstock may include bioprocessessuch as saccharifying and/or fermenting the feedstock, e.g., bycontacting the pretreated material with an enzyme and/or microorganism.Products produced by such contact can include any of those productsdescribed herein, such as food or fuel, e.g., ethanol, or any otherproducts described in U.S. Provisional Application Ser. No. 61/139,453.

Fermentation

Generally, various microorganisms can produce a number of usefulproducts, such as a fuel, by operating on, e.g., fermenting thepretreated biomass materials. For example, alcohols, organic acids,hydrocarbons, hydrogen, proteins or mixtures of any of these materialscan be produced by fermentation or other bioprocesses.

The microorganism can be a natural microorganism or an engineeredmicroorganism. For example, the microorganism can be a bacterium, e.g.,a cellulolytic bacterium, a fungus, e.g., a yeast, a plant or a protist,e.g., an algae, a protozoa or a fungus-like protist, e.g., a slime mold.When the organisms are compatible, mixtures of organisms can beutilized.

To aid in the breakdown of the materials that include the cellulose, oneor more enzymes, e.g., a cellulolytic enzyme can be utilized. In someembodiments, the materials that include the cellulose are first treatedwith the enzyme, e.g., by combining the material and the enzyme in anaqueous solution. This material can then be combined with themicroorganism. In other embodiments, the materials that include thecellulose, the one or more enzymes and the microorganism are combinedconcurrently, e.g., by combining in an aqueous solution.

The pretreated material can be treated with heat and/or a chemical(e.g., mineral acid, base or a strong oxidizer such as sodiumhypochlorite) to further facilitate breakdown.

During fermentation, sugars released from cellulolytic hydrolysis orsaccharification are fermented to, e.g., ethanol, by a fermentingmicroorganism such as yeast. Suitable fermenting microorganisms have theability to convert carbohydrates, such as glucose, xylose, arabinose,mannose, galactose, oligosaccharides or polysaccharides intofermentation products. Fermenting microorganisms include strains of thegenus Sacchromyces spp. e.g., Sacchromyces cerevisiae (baker's yeast),Saccharomyces distaticus, Saccharomyces uvarum; the genus Kluyveromyces,e.g., species Kluyveromyces marxianus, Kluyveromyces fragilis; the genusCandida, e.g., Candida pseudotropicalis, and Candida brassicae, thegenus Clavispora, e.g., species Clavispora lusitaniae and Clavisporaopuntiae the genus Pachysolen, e.g., species Pachysolen tannophilus, thegenus Bretannomyces, e.g., species Bretannomyces clausenii (Philippidis,G. P., 1996, Cellulose bioconversion technology, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212).

Commercially available yeasts include, for example, Red Star®/LesaffreEthanol Red (available from Red Star/Lesaffre, USA) FALI® (availablefrom Fleischmann's Yeast, a division of Burns Philip Food Inc., USA),SUPERSTART® (available from Alltech), GERT STRAND® (available from GertStrand AB, Sweden) and FERMOL® (available from DSM Specialties).

Bacteria that can ferment biomass to ethanol and other products include,e.g., Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996,supra). Leschine et al. (International Journal of Systematic andEvolutionary Microbiology 2002, 52, 1155-1160) isolated an anaerobic,mesophilic, cellulolytic bacterium from forest soil, Clostridiumphytofermentans sp. nov., which converts cellulose to ethanol.

Fermentation of biomass to ethanol and other products may be carried outusing certain types of thermophilic or genetically engineeredmicroorganisms, such Thermoanaerobacter species, including T. mathranii,and yeast species such as Pichia species. An example of a strain of T.mathranii is A3M4 described in Sonne-Hansen et al. (Applied Microbiologyand Biotechnology 1993, 38, 537-541) or Ahring et al. (Arch. Microbiol.1997, 168, 114-119).

Yeast and Zymomonas bacteria can be used for fermentation or conversion.The optimum pH for yeast is from about pH 4 to 5, while the optimum pHfor Zymomonas is from about pH 5 to 6. Typical fermentation times areabout 24 to 96 hours with temperatures in the range of 26° C. to 40° C.,however thermophilic microorganisms prefer higher temperatures.

Enzymes and biomass-destroying organisms that break down biomass, suchas the cellulose and/or the lignin portions of the biomass, to lowermolecular weight of the carbohydrate-containing materials contain ormake various cellulolytic enzymes (cellulases), ligninases or varioussmall molecule biomass-destroying metabolites. These enzymes may be acomplex of enzymes that act synergistically to degrade crystallinecellulose or the lignin portions of biomass. Examples of cellulolyticenzymes include: endoglucanases, cellobiohydrolases, and cellobiases(β-glucosidases). A cellulosic substrate is initially hydrolyzed byendoglucanases at random locations producing oligomeric intermediates.These intermediates are then substrates for exo-splitting glucanasessuch as cellobiohydrolase to produce cellobiose from the ends of thecellulose polymer. Cellobiose is a water-soluble 1,4-linked dimer ofglucose. Finally cellobiase cleaves cellobiose to yield glucose.

A cellulase is capable of degrading biomass and may be of fungal orbacterial origin. Suitable enzymes include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,Chrysosporium and Trichoderma, and include species of Humicola,Coprinus, Thielavia, Fusarium, Myceliophthora, Acremonium,Cephalosporium, Scytalidium, Penicillium or Aspergillus (see, e.g., EP458162), especially those produced by a strain selected from the speciesHumicola insolens (reclassified as Scytalidium thermophilum, see, e.g.,U.S. Pat. No. 4,435,307), Coprinus cinereus, Fusarium oxysporum,Myceliophthora thermophile, Meripilus giganteus, Thielavia terrestris,Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremoniumbrachypenium, Acremonium dichromosporum, Acremonium obclavatum,Acremonium pinkertoniae, Acremonium roseogriseum, Acremoniumincoloratum, and Acremonium furatum; preferably from the speciesHumicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthorathermophila CBS 117.65, Cephalosporium sp. RYM-202, Acremonium sp. CBS478.94, Acremonium sp. CBS 265.95, Acremonium persicinum CBS 169.65,Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.71,Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae CBS157.70, Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS146.62, and Acremonium furatum CBS 299.70H. Cellulolytic enzymes mayalso be obtained from Chrysosporium, preferably a strain ofChrysosporium lucknowense. Additionally, Trichoderma (particularlyTrichoderma viride, Trichoderma reesei, and Trichoderma koningii),alkalophilic Bacillus (see, for example, U.S. Pat. No. 3,844,890 and EP458162), and Streptomyces (see, e.g., EP 458162) may be used.

Anaerobic cellulolytic bacteria have also been isolated from soil, e.g.,a novel cellulolytic species of Clostridium, Clostridium phytofermentanssp. nov. (see Leschine et. al, International Journal of Systematic andEvolutionary Microbiology (2002), 52, 1155-1160).

Cellulolytic enzymes using recombinant technology can also be used (see,e.g., WO 2007/071818 and WO 2006/110891).

The cellulolytic enzymes used can be produced by fermentation of theabove-noted microbial strains on a nutrient medium containing suitablecarbon and nitrogen sources and inorganic salts, using procedures knownin the art (see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, CA 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand cellulase production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, NY, 1986).

Treatment of cellulose with cellulase is usually carried out attemperatures between 30° C. and 65° C. Cellulases are active over arange of pH of about 3 to 7. A saccharification step may last up to 120hours. The cellulase enzyme dosage achieves a sufficiently high level ofcellulose conversion. For example, an appropriate cellulase dosage istypically between 5.0 and 50 Filter Paper Units (FPU or IU) per gram ofcellulose. The FPU is a standard measurement and is defined and measuredaccording to Ghose (1987, Pure and Appl. Chem. 59:257-268).

Mobile fermentors can be utilized, as described in U.S. ProvisionalPatent Application Ser. 60/832,735, now Published InternationalApplication No. WO 2008/011598.

Products/Co-Products

Using such primary processes and/or post-processing, the treated biomasscan be converted to one or more products, for example alcohols, e.g.,methanol, ethanol, propanol, isopropanol, butanol, e.g., n-, sec- ort-butanol, ethylene glycol, propylene glycol, 1,4-butane diol, glycerinor mixtures of these alcohols; organic acids, such as formic acid,acetic acid, propionic acid, butyric acid, valeric acid, caproic,palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid,glutaric acid, oleic acid, linoleic acid, glycolic acid, lactic acid,γ-hydroxybutyric acid or mixtures of these acids; food products; animalfeed; pharmaceuticals; or nutriceuticals. Co-products that may beproduced include lignin residue.

EXAMPLES

The following Examples are intended to illustrate, and do not limit theteachings of this disclosure.

Example 1 Preparation of Fibrous Material from Polycoated Paper

A 1500 pound skid of virgin, half-gallon juice cartons made ofun-printed polycoated white Kraft board having a bulk density of 20lb/ft³ was obtained from International Paper. Each carton was foldedflat, and then fed into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder was equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades was adjusted to 0.10inch. The output from the shredder resembled confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inchand a thickness equivalent to that of the starting material (about 0.075inch).

The confetti-like material was fed to a Munson rotary knife cutter,Model SC30. Model SC30 is equipped with four rotary blades, four fixedblades, and a discharge screen having ⅛ inch openings. The gap betweenthe rotary and fixed blades was set to approximately 0.020 inch. Therotary knife cutter sheared the confetti-like pieces across theknife-edges, tearing the pieces apart and releasing a fibrous materialat a rate of about one pound per hour. The fibrous material had a BETsurface area of 0.9748 m²/g+/−0.0167 m²/g, a porosity of 89.0437 percentand a bulk density (@0.53 psia) of 0.1260 g/mL. An average length of thefibers was 1.141 mm and an average width of the fibers was 0.027 mm,giving an average L/D of 42:1. A scanning electron micrograph of thefibrous material is shown in FIG. 9 at 25× magnification.

Example 2 Preparation of Fibrous Material from Bleached Kraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch and a thickness equivalent to that of the startingmaterial (about 0.075 inch). The confetti-like material was fed to aMunson rotary knife cutter, Model SC30. The discharge screen had ⅛ inchopenings. The gap between the rotary and fixed blades was set toapproximately 0.020 inch. The rotary knife cutter sheared theconfetti-like pieces, releasing a fibrous material at a rate of aboutone pound per hour. The fibrous material had a BET surface area of1.1316 m²/g+/−0.0103 m²/g, a porosity of 88.3285 percent and a bulkdensity (@0.53 psia) of 0.1497 g/mL. An average length of the fibers was1.063 mm and an average width of the fibers was 0.0245 mm, giving anaverage L/D of 43:1. A scanning electron micrographs of the fibrousmaterial is shown in FIG. 10 at 25× magnification.

Example 3 Preparation of Twice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had 1/16 inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. The material resultingfrom the first shearing was fed back into the same setup described aboveand sheared again. The resulting fibrous material had a BET surface areaof 1.4408 m²/g+/−0.0156 m²/g, a porosity of 90.8998 percent and a bulkdensity (@0.53 psia) of 0.1298 g/mL. An average length of the fibers was0.891 mm and an average width of the fibers was 0.026 mm, giving anaverage L/D of 34:1. A scanning electron micrograph of the fibrousmaterial is shown in FIG. 11 at 25× magnification.

Example 4 Preparation of Thrice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had ⅛ inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter sheared the confetti-like pieces across theknife-edges. The material resulting from the first shearing was fed backinto the same setup and the screen was replaced with a 1/16 inch screen.This material was sheared. The material resulting from the secondshearing was fed back into the same setup and the screen was replacedwith a 1/32 inch screen. This material was sheared. The resultingfibrous material had a BET surface area of 1.6897 m²/g+/−0.0155 m²/g, aporosity of 87.7163 percent and a bulk density (@0.53 psia) of 0.1448g/mL. An average length of the fibers was 0.824 mm and an average widthof the fibers was 0.0262 mm, giving an average L/D of 32:1. A scanningelectron micrograph of the fibrous material is shown in FIG. 12 at 25×magnification.

Example 5 Methods of Determining Molecular Weight of Cellulosic andLignocellulosic Materials by Gel Permeation Chromatography

Cellulosic and lignocellulosic materials for analysis were treatedaccording to Example 4. Sample materials presented in the followingtables include Kraft paper (P), wheat straw (WS), alfalfa (A), andswitchgrass (SG). The number “132” of the Sample ID refers to theparticle size of the material after shearing through a 1/32 inch screen.The number after the dash refers to the dosage of radiation (MRad) and“US” refers to ultrasonic treatment. For example, a sample ID “P132-10”refers to Kraft paper that has been sheared to a particle size of 132mesh and has been irradiated with 10 MRad.

TABLE 1 Peak Average Molecular Weight of Irradiated Kraft Paper SampleDosage¹ Average MW ± Source Sample ID (MRad) Ultrasound² Std Dev. KraftPaper P132 0 No 32853 ± 10006 P132-10 10 ″  61398 ± 2468** P132-100 100″ 8444 ± 580  P132-181 181 ″ 6668 ± 77  P132-US 0 Yes 3095 ± 1013 **Lowdoses of radiation appear to increase the molecular weight of somematerials ¹Dosage Rate = 1 MRad/hour ²Treatment for 30 minutes with 20kHz ultrasound using a 1000 W horn under re-circulating conditions withthe material dispersed in water.

TABLE 2 Peak Average Molecular Weight of Irradiated Materials Dosage¹Average MW ± Sample ID Peak # (MRad) Ultrasound² Std Dev. WS132 1  0 No1407411 ± 175191 2 ″ ″ 39145 ± 3425 3 ″ ″ 2886 ± 177 WS132-10* 1  10 ″26040 ± 3240 WS132-100* 1 100 ″ 23620 ± 453  A132 1  0 ″ 1604886 ±151701 2 ″ ″ 37525 ± 3751 3 ″ ″ 2853 ± 490 A132-10* 1  10 ″ 50853 ± 16652 ″ ″ 2461 ± 17  A132-100* 1 100 ″ 38291 ± 2235 2 ″ ″ 2487 ± 15  SG132 1 0 ″ 1557360 ± 83693  2 ″ ″ 42594 ± 4414 3 ″ ″ 3268 ± 249 SG132-10* 1 10 ″ 60888 ± 9131 SG132-100* 1 100 ″ 22345 ± 3797 SG132-10-US 1  10 Yes 86086 ± 43518 2 ″ ″ 2247 ± 468 SG132-100-US 1 100 ″  4696 ± 1465 *Peakscoalesce after treatment **Low doses of radiation appear to increase themolecular weight of some materials ¹Dosage Rate = 1 MRad/hour ²Treatmentfor 30 minutes with 20 kHz ultrasound using a 1000 W horn underre-circulating conditions with the material dispersed in water.

Gel Permeation Chromatography (GPC) is used to determine the molecularweight distribution of polymers. During GPC analysis, a solution of thepolymer sample is passed through a column packed with a porous geltrapping small molecules. The sample is separated based on molecularsize with larger molecules eluting sooner than smaller molecules. Theretention time of each component is most often detected by refractiveindex (RI), evaporative light scattering (ELS), or ultraviolet (UV) andcompared to a calibration curve. The resulting data is then used tocalculate the molecular weight distribution for the sample.

A distribution of molecular weights rather than a unique molecularweight is used to characterize synthetic polymers. To characterize thisdistribution, statistical averages are utilized. The most common ofthese averages are the “number average molecular weight” (M_(n)) and the“weight average molecular weight” (MO.

M_(n) is similar to the standard arithmetic mean associated with a groupof numbers. When applied to polymers, M_(n) refers to the averagemolecular weight of the molecules in the polymer. M_(n) is calculatedaffording the same amount of significance to each molecule regardless ofits individual molecular weight. The average M_(n) is calculated by thefollowing formula where N_(i) is the number of molecules with a molarmass equal to

${\overset{\_}{M}}_{n} = \frac{\sum\limits_{i}{N_{i}M_{i}}}{\sum\limits_{i}N_{i}}$

M_(w) is another statistical descriptor of the molecular weightdistribution that places a greater emphasis on larger molecules thansmaller molecules in the distribution. The formula below shows thestatistical calculation of the weight average molecular weight.

${\overset{\_}{M}}_{w} = \frac{\sum\limits_{i}{N_{i}M_{i}^{2}}}{\sum\limits_{i}{N_{i}M_{i}}}$

The polydispersity index or PI is defined as the ratio of M_(W)/M_(n).The larger the PI, the broader or more disperse the distribution. Thelowest value that a PI can be is 1. This represents a monodispersesample; that is, a polymer with all of the molecules in the distributionbeing the same molecular weight.

The peak molecular weight value (M_(P)) is another descriptor defined asthe mode of the molecular weight distribution. It signifies themolecular weight that is most abundant in the distribution. This valuealso gives insight to the molecular weight distribution.

Most GPC measurements are made relative to a different polymer standard.The accuracy of the results depends on how closely the characteristicsof the polymer being analyzed match those of the standard used. Theexpected error in reproducibility between different series ofdeterminations, calibrated separately, is ca. 5-10% and ischaracteristic to the limited precision of GPC determinations.Therefore, GPC results are most useful when a comparison between themolecular weight distribution of different samples is made during thesame series of determinations.

The lignocellulosic samples required sample preparation prior to GPCanalysis. First, a saturated solution (8.4% by weight) of lithiumchloride (LiCl) was prepared in dimethyl acetamide (DMAc). Approximately100 mg of the sample was added to approximately 10 g of a freshlyprepared saturated LiCl/DMAc solution, and the mixture was heated toapproximately 150° C.-170° C. with stirring for 1 hour. The resultingsolutions were generally light- to dark-yellow in color. The temperatureof the solutions were decreased to approximately 100° C. and heated foran additional 2 hours. The temperature of the solutions were thendecreased to approximately 50° C. and the sample solution was heated forapproximately 48 to 60 hours. Of note, samples irradiated at 100 MRadwere more easily solubilized as compared to their untreated counterpart.Additionally, the sheared samples (denoted by the number 132) hadslightly lower average molecular weights as compared with uncut samples.

The resulting sample solutions were diluted 1:1 using DMAc as solventand were filtered through a 0.45 μm PTFE filter. The filtered samplesolutions were then analyzed by GPC. The peak average molecular weight(Mp) of the samples, as determined by Gel Permeation Chromatography(GPC), are summarized in Tables 1 and 2. Each sample was prepared induplicate and each preparation of the sample was analyzed in duplicate(two injections) for a total of four injections per sample. The EasiCal®polystyrene standards PS1A and PS1B were used to generate a calibrationcurve for the molecular weight scale from about 580 to 7,500,00 Daltons.Table 3 recites the GPC analysis conditions.

TABLE 3 GPC Analysis Conditions Instrument: Waters Alliance GPC 2000Plgel 10μ Mixed-B Columns (3): S/N's: 10M-MB-148-83; 10M-MB- 148-84;10M-MB-174-129 Mobile Phase (solvent): 0.5% LiCl in DMAc (1.0 mL/min.)Column/Detector Temperature: 70° C. Injector Temperature: 70° C. SampleLoop Size: 323.5 μL

Example 6 Porosimetry Analysis of Irradiated Materials

Mercury pore size and pore volume analysis (Table 4) is based on forcingmercury (a non-wetting liquid) into a porous structure under tightlycontrolled pressures. Since mercury does not wet most substances andwill not spontaneously penetrate pores by capillary action, it must beforced into the voids of the sample by applying external pressure. Thepressure required to fill the voids is inversely proportional to thesize of the pores. Only a small amount of force or pressure is requiredto fill large voids, whereas much greater pressure is required to fillvoids of very small pores.

TABLE 4 Pore Size and Volume Distribution by Mercury Porosimetry MedianMedian Average Bulk Total Total Pore Pore Pore Density ApparentIntrusion Pore Diameter Diameter Diameter @ 0.50 (skeletal) Volume Area(Volume) (Area) (4 V/A) psia Density Porosity Sample ID (mL/g) (m²/g)(μm) (μm) (μm) (g/mL) (g/mL) (%) P132 6.0594 1.228 36.2250 13.727819.7415 0.1448 1.1785 87.7163 P132-10 5.5436 1.211 46.3463 4.564618.3106 0.1614 1.5355 89.4875 P132-100 5.3985 0.998 34.5235 18.200521.6422 0.1612 1.2413 87.0151 P132-181 3.2866 0.868 25.3448 12.241015.1509 0.2497 1.3916 82.0577 P132-US 6.0005 14.787 98.3459 0.00551.6231 0.1404 0.8894 84.2199 A132 2.0037 11.759 64.6308 0.0113 0.68160.3683 1.4058 73.7990 A132-10 1.9514 10.326 53.2706 0.0105 0.7560 0.37681.4231 73.5241 A132-100 1.9394 10.205 43.8966 0.0118 0.7602 0.37601.3889 72.9264 SG132 2.5267 8.265 57.6958 0.0141 1.2229 0.3119 1.470878.7961 SG132-10 2.1414 8.643 26.4666 0.0103 0.9910 0.3457 1.331574.0340 SG132-100 2.5142 10.766 32.7118 0.0098 0.9342 0.3077 1.359077.3593 SG132-10-US 4.4043 1.722 71.5734 1.1016 10.2319 0.1930 1.288385.0169 SG132-100-US 4.9665 7.358 24.8462 0.0089 2.6998 0.1695 1.073184.2010 WS132 2.9920 5.447 76.3675 0.0516 2.1971 0.2773 1.6279 82.9664WS132-10 3.1138 2.901 57.4727 0.3630 4.2940 0.2763 1.9808 86.0484WS132-100 3.2077 3.114 52.3284 0.2876 4.1199 0.2599 1.5611 83.3538

The AutoPore® 9520 can attain a maximum pressure of 414 MPa or 60,000psia. There are four low pressure stations for sample preparation andcollection of macropore data from 0.2 psia to 50 psia. There are twohigh pressure chambers which collects data from 25 psia to 60,000 psia.The sample is placed in a bowl-like apparatus called a penetrometer,which is bonded to a glass capillary stem with a metal coating. Asmercury invades the voids in and around the sample, it moves down thecapillary stem. The loss of mercury from the capillary stem results in achange in the electrical capacitance. The change in capacitance duringthe experiment is converted to volume of mercury by knowing the stemvolume of the penetrometer in use. A variety of penetrometers withdifferent bowl (sample) sizes and capillaries are available toaccommodate most sample sizes and configurations. Table 5 below definessome of the key parameters calculated for each sample.

TABLE 5 Definition of Parameters Parameter Description Total IntrusionVolume: The total volume of mercury intruded during an experiment. Thiscan include interstitial filling between small particles, porosity ofsample, and compression volume of sample. Total Pore Area: The totalintrusion volume converted to an area assuming cylindrical shaped pores.Median Pore Diameter The size at the 50^(th) percentile (volume): on thecumulative volume graph. Median Pore Diameter (area): The size at the50^(th) percentile on the cumulative area graph. Average Pore Diameter:The total pore volume divided by the total pore area (4 V/A). BulkDensity: The mass of the sample divided by the bulk volume. Bulk volumeis determined at the filling pressure, typically 0.5 psia. ApparentDensity: The mass of sample divided by the volume of sample measured athighest pressure, typically 60,000 psia. Porosity: (BulkDensity/Apparent Density) × 100%

Example 7 Particle Size Analysis of Irradiated Materials

The technique of particle sizing by static light scattering is based onMie theory (which also encompasses Fraunhofer theory). Mie theorypredicts the intensity vs. angle relationship as a function of the sizefor spherical scattering particles provided that other system variablesare known and held constant. These variables are the wavelength ofincident light and the relative refractive index of the sample material.Application of Mie theory provides the detailed particle sizeinformation. Table 6 summarizes particle size using median diameter,mean diameter, and modal diameter as parameters.

TABLE 6 Particle Size by Laser Light Scattering (Dry Sample Dispersion)Median Diameter Mean Diameter Modal Diameter Sample ID (μm) (μm) (μm)A132 380.695 418.778 442.258 A132-10 321.742 366.231 410.156 A132-100301.786 348.633 444.169 SG132 369.400 411.790 455.508 SG132-10 278.793325.497 426.717 SG132-100 242.757 298.686 390.097 WS132 407.335 445.618467.978 WS132-10 194.237 226.604 297.941 WS132-100 201.975 236.037307.304

Particle size was determined by Laser Light Scattering (Dry SampleDispersion) using a Malvern Mastersizer 2000 using the followingconditions:

Feed Rate: 35% Disperser Pressure: 4 Bar Optical Model: (2.610, 1.000i),1.000

An appropriate amount of sample was introduced onto a vibratory tray.The feed rate and air pressure were adjusted to ensure that theparticles were properly dispersed. The key component is selecting an airpressure that will break up agglomerations, but does not compromise thesample integrity. The amount of sample needed varies depending on thesize of the particles. In general, samples with fine particles requireless material than samples with coarse particles.

Example 8 Surface Area Analysis of Irradiated Materials

Surface area of each sample was analyzed using a Micromeritics® ASAP2420 Accelerated Surface Area and Porosimetry System. The samples wereprepared by first degassing for 16 hours at 40° C. Next, free space(both warm and cold) with helium is calculated and then the sample tubeis evacuated again to remove the helium. Data collection begins afterthis second evacuation and consists of defining target pressures whichcontrols how much gas is dosed onto the sample. At each target pressure,the quantity of gas adsorbed and the actual pressure are determined andrecorded. The pressure inside the sample tube is measured with apressure transducer. Additional doses of gas will continue until thetarget pressure is achieved and allowed to equilibrate. The quantity ofgas adsorbed is determined by summing multiple doses onto the sample.The pressure and quantity define a gas adsorption isotherm and are usedto calculate a number of parameters, including BET surface area (Table7).

TABLE 7 Summary of Surface Area by Gas Adsorption BET Surface Sample IDSingle point surface area (m²/g) Area (m²/g) P132 @ P/Po = 0.2503877711.5253 1.6897 P132-10 @ P/Po = 0.239496722 1.0212 1.2782 P132-100 @ P/Po= 0.240538100 1.0338 1.2622 P132-181 @ P/Po = 0.239166091 0.5102 0.6448P132-US @ P/Po = 0.217359072 1.0983 1.6793 A132 @ P/Po = 0.2400406100.5400 0.7614 A132-10 @ P/Po = 0.211218936 0.5383 0.7212 A132-100 @ P/Po= 0.238791097 0.4258 0.5538 SG132 @ P/Po = 0.237989353 0.6359 0.8350SG132-10 @ P/Po = 0.238576905 0.6794 0.8689 SG132-100 @ P/Po =0.241960361 0.5518 0.7034 SG132-10-US @ P/Po = 0.225692889 0.5693 0.7510SG132-100-US @ P/Po = 0.225935246 1.0983 1.4963 WS132 @ P/Po =0.237823664 0.6582 0.8663 WS132-10 @ P/Po = 0.238612476 0.6191 0.7912WS132-100 @ P/Po = 0.238398832 0.6255 0.8143

The BET model for isotherms is a widely used theory for calculating thespecific surface area. The analysis involves determining the monolayercapacity of the sample surface by calculating the amount required tocover the entire surface with a single densely packed layer of krypton.The monolayer capacity is multiplied by the cross sectional area of amolecule of probe gas to determine the total surface area. Specificsurface area is the surface area of the sample aliquot divided by themass of the sample.

Example 9 Fiber Length Determination of Irradiated Materials

Fiber length distribution testing was performed in triplicate on thesamples submitted using the Techpap MorFi LB01 system. The averagelength and width are reported in Table 8.

TABLE 8 Summary of Lignocellulosic Fiber Length and Width Data AverageStatistically Length Corrected Arithmetic Weighted Average Length WidthAverage in Length Weighted in (micrometers) Sample ID (mm) (mm) Length(mm) (μm) P132-10 0.484 0.615 0.773 24.7 P132-100 0.369 0.423 0.496 23.8P132-181 0.312 0.342 0.392 24.4 A132-10 0.382 0.423 0.650 43.2 A132-1000.362 0.435 0.592 29.9 SG132-10 0.328 0.363 0.521 44.0 SG132-100 0.3250.351 0.466 43.8 WS132-10 0.353 0.381 0.565 44.7 WS132-100 0.354 0.3710.536 45.4

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

Lignases and Biomass Destroying Enzymes

For example, some methods utilize one or more ligninases and/orbiomass-destroying enzymes, instead of or in addition to Fentonchemistry, to reduce recalcitrance in cellulosic or lignocellulosicmaterials. In such methods, a first cellulosic or lignocellulosicmaterial having a first level of recalcitrance is provided and combinedwith one or more ligninases and/or one or more biomass-destroying, e.g.,lignin-destroying organisms, so as to contact the first cellulosic orlignocellulosic material. The contact is maintained for a period oftime, such as between 2 and 24 hours, e.g., between 6 and 12 hours, andunder conditions sufficient, e.g., below a pH of about 6, such asbetween pH 3 and 5.5, to produce a second lignocellulosic materialhaving a second level of recalcitrance lower than the first level ofrecalcitrance. After reduction of the recalcitrance, the secondcellulosic or lignocellulosic material can be contacted with one or moreenzymes and/or microorganisms, e.g., to make any product describedherein, e.g., food or fuel, e.g., ethanol or butanol (e.g., n-butanol)or any product described in any application incorporated by referenceherein.

The ligninase can be, e.g., one or more of manganese peroxidase, ligninperoxidase or laccases.

In particular implementations, the biomass-destroying organism can be,e.g., one or more of white rot, brown rot or soft rot. For example, thebiomass-destroying organism can be a Basidiomycetes fungus. Inparticular embodiments, the biomass-destroying organism Phanerochaetechrysoporium or Gleophyllum trabeum.

Ligninases, biomass-destroying organisms and small molecule metabolitesare described in Kirk et al., Enzyme Microb. Technol. 1986, vol. 8,27-32, Kirk et al., Enzymes for Pulp and Paper Processing, Chapter 1(Roles for Microbial Enzymes in Pulp and Paper Processing and Kirk etal., The Chemistry of Solid Wood, Chapter 12 (Biological Decompositionof Solid Wood (pp. 455-487).

Hydrocarbon-Containing Materials

In some embodiments, the methods and systems disclosed herein can beused to process hydrocarbon-containing materials such as tar or oilsands, oil shale, crude oil (e.g., heavy crude oil and/or light crudeoil), bitumen, coal, petroleum gases (e.g., methane, ethane, propane,butane, isobutane), liquefied natural and/or synthetic gas, asphalt, andother natural materials that include various types of hydrocarbons. Forexample, a processing facility for hydrocarbon-containing materialsreceives a supply of raw material. The raw material can be delivereddirectly from a mine, e.g., by conveyor belt and/or rail car system, andin certain embodiments, the processing facility can be constructed inrelatively close proximity to, or even atop, the mine. In someembodiments, the raw material can be transported to the processingfacility via railway freight car or another motorized transport system,and/or pumped to the processing facility via pipeline.

When the raw material enters the processing facility, the raw materialcan be broken down mechanically and/or chemically to yield startingmaterial. As an example, the raw material can include material derivedfrom oil sands and containing crude bitumen. Bitumen can then beprocessed into one or more hydrocarbon products using the methodsdisclosed herein. In some embodiments, the oil sands material can beextracted from surface mines such as open pit mines. In certainembodiments, sub-surface oil sands material can be extracted using a hotwater flotation process that removes oil from sand particles, and thenadding naphtha to allow pumping of the oil to the processing facility.

Bitumen processing generally includes two stages. In a first stage,relatively large bitumen hydrocarbons are cracked into smaller moleculesusing coking, hydrocracking, or a combination of the two techniques. Inthe coking process, carbon is removed from bitumen hydrocarbon moleculesat high temperatures (e.g., 400° C. or more), leading to cracking of themolecules. In hydrocracking, hydrogen is added to bitumen molecules,which are then cracked over a catalyst system (e.g., platinum).

In a second stage, the cracked bitumen molecules are hydrotreated. Ingeneral, hydrotreating includes heating the cracked bitumen molecules ina hydrogen atmosphere to remove metals, nitrogen (e.g., as ammonia), andsulfur (e.g., as elemental sulfur).

The overall bitumen processing procedure typically producesapproximately one barrel of synthetic crude oil for every 2.5 tons ofoil sand material processed. Moreover, an energy equivalent ofapproximately one barrel of oil is used to produce three barrels ofsynthetic crude oil from oil sand-derived bitumen sources.

As another example, oil shale typically includes fine-grainedsedimentary rock that includes significant amounts of kerogen (a mixtureof various organic compounds in solid form). By heating oil shale, avapor is liberated which can be purified to yield a hydrocarbon richshale oil and a combustible hydrocarbon shale gas. Typically, the oilshale is heated to between 250° C. and 550° C. in the absence of oxygento liberate the vapor.

The efficiency and cost-effectiveness with which usable hydrocarbonproducts can be extracted from oil sands material, oil shale, crude oil,and other oil-based raw materials can be improved by applying themethods disclosed herein. In addition, a variety of differenthydrocarbon products (including various hydrocarbon fractions that arepresent in the raw material, and other types of hydrocarbons that areformed during processing) can be extracted from the raw materials.

In certain embodiments, in addition to Fenten oxidation, other methodscan also be used to process raw and/or intermediatehydrocarbon-containing materials. For example, electron beams or ionbeams can be used to process the materials. For example, ion beams thatinclude one or more different types of ions (e.g., protons, carbon ions,oxygen ions, hydride ions) can be used to process raw materials. The ionbeams can include positive ions and/or negative ions, in doses that varyfrom 1 Mrad to 2500 Mrad or more, e.g., 50, 100, 250, 350, 500, 1000,1500, 2000, or 2500 MRad, or even higher levels.

Other additional processing methods can be used, including oxidation,pyrolysis, and sonication. In general, process parameters for each ofthese techniques when treating hydrocarbon-based raw and/or intermediatematerials can be the same as those disclosed above in connection withbiomass materials. Various combinations of these techniques can also beused to process raw or intermediate materials.

Generally, the various techniques can be used in any order, and anynumber of times, to treat raw and/or intermediate materials. Forexample, to process bitumen from oil sands, one or more of thetechniques disclosed herein can be used prior to any mechanicalbreakdown steps, following one or more mechanical breakdown steps, priorto cracking, after cracking and/or prior to hydrotreatment, and afterhydrotreatment. As another example, to process oil shale, one or more ofthe techniques disclosed herein can be used prior to either or both ofthe vaporization and purification steps discussed above. Productsderived from the hydrocarbon-based raw materials can be treated againwith any combination of techniques prior to transporting the productsout of the processing facility (e.g., either via motorized transport, orvia pipeline).

The techniques disclosed herein can be applied to process raw and/orintermediate material in dry form, in a solution or slurry, or ingaseous form (e.g., to process hydrocarbon vapors at elevatedtemperature). The solubility of raw or intermediate products insolutions and slurries can be controlled through selective addition ofone or more agents such as acids, bases, oxidizing agents, reducingagents, and salts. In general, the methods disclosed herein can be usedto initiate and/or sustain the reaction of raw and/or intermediatehydrocarbon-containing materials, extraction of intermediate materialsfrom raw materials (e.g., extraction of hydrocarbon components fromother solid or liquid components), distribution of raw and/orintermediate materials, and separation of intermediate materials fromraw materials (e.g., separation of hydrocarbon-containing componentsfrom other solid matrix components to increase the concentration and/orpurity and/or homogeneity of the hydrocarbon components).

In addition, microorganisms can be used for processing raw orintermediate materials, either prior to or following the use of themethods described herein. Suitable microorganisms include various typesof bacteria, yeasts, and mixtures thereof, as disclosed previously. Theprocessing facility can be equipped to remove harmful byproducts thatresult from the processing of raw or intermediate materials, includinggaseous products that are harmful to human operators, and chemicalbyproducts that are harmful to humans and/or various microorganisms.

In some embodiments, the use of one or more of the techniques disclosedherein results in a molecular weight reduction of one or more componentsof the raw or intermediate material that is processed. As a result,various lower weight hydrocarbon substances can be produced from one ormore higher weight hydrocarbon substances. In certain embodiments, theuse of one or more of the techniques disclosed herein results in anincrease in molecular weight of one or more components of the raw orintermediate material that is processed. For example, the varioustechniques disclosed herein can induce bond-formation between moleculesof the components, leading to the formation of increased quantities ofcertain products, and even to new, higher molecular weight products. Inaddition to hydrocarbon products, various other compounds can beextracted from the raw materials, including nitrogen based compounds(e.g., ammonia), sulfur-based compounds, and silicates and othersilicon-based compounds. In certain embodiments, one or more productsextracted from the raw materials can be combusted to generate processheat for heating water, raw or intermediate materials, generatingelectrical power, or for other applications.

Processing oil sand materials (including bitumen) using one or more ofthe techniques disclosed herein can lead to more efficient crackingand/or hydrotreatment of the bitumen. As another example, processing oilshale can lead to more efficient extraction of various products,including shale oil and/or shale gas, from the oil shale. In certainembodiments, steps such as cracking or vaporization may not even benecessary if the techniques disclosed herein are first used to treat theraw material. Further, in some embodiments, by treating raw and/orintermediate materials, the products can be made more soluble in certainsolvents, in preparation for subsequent processing steps in solution(e.g., steam blasting, sonication). Improving the solubility of theproducts can improve the efficiency of subsequent solution-basedtreatment steps. By improving the efficiency of other processing steps(e.g., cracking and/or hydrotreatment of bitumen, vaporization of oilshale), the overall energy consumed in processing the raw materials canbe reduced, making extraction and processing of the raw materialseconomically feasible.

Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of reducing recalcitrance in cellulosic or lignocellulosicmaterials, the method comprising: contacting, in a mixture, a firstcellulosic or lignocellulosic material having a first level ofrecalcitrance with one or more compounds comprising one or morenaturally-occurring, non-radioactive group 5, 6, 7, 8, 9, 10 or 11elements, to produce a second cellulosic or lignocellulosic materialhaving a second level of recalcitrance lower than the first level ofrecalcitrance.
 2. The method of claim 1, wherein the one or moreelements are in a 1+, 2+, 3+, 4+ or 5+ oxidation state.
 3. The method ofclaim 1, wherein the one or more elements comprise Mn, Fe, Co, Ni, Cu orZn.
 4. The method of claim 1, wherein the one or more elements compriseFe in the 2+, 3+ or 4+ oxidation state.
 5. The method of claim 1,wherein the mixture further comprises one or more oxidants capable ofincreasing an oxidation state of at least some of said elements.
 6. Themethod of claim 5 in which the oxidant comprises ozone and/or hydrogenperoxide.
 7. The method of claim 1 further comprising maintaining pH ator below about 5.5 during contact.
 8. The method of claim 5 furthercomprising dispersing the first cellulosic or lignocellulosic materialin water or an aqueous medium, and then adding first the one or morecompounds and then the one or more oxidants.
 9. The method of claim 5further comprising dispersing the first cellulosic or lignocellulosicmaterial in water or an aqueous medium, and then adding first the one ormore oxidants and then the one or more compounds.
 10. The method ofclaim 1, wherein a total maximum concentration of the elements in theone or more compounds measured in the dispersion is from about 10 μM toabout 500 mM.
 11. The method of claim 5, wherein a total maximumconcentration of the one or more oxidants is from about 100 μM to about1 M.
 12. The method of claim 5 in which the one or more oxidants areapplied to the first cellulosic or lignocellulosic material and the oneor more compounds as a gas, such as by generating ozone in-situ byirradiating the first cellulosic or lignocellulosic and the one or morecompounds through air with a beam of particles
 13. The method of claim12 wherein the particles are selected from the group consisting ofelectrons and protons.
 14. The method of claim 1 wherein the mixtureincludes one or more compounds and one or more oxidants, and wherein amole ratio of the element(s) in the one or more compounds to the one ormore oxidants is from about 1:1000 to about 1:25.
 15. The method ofclaim 1, wherein the mixture further includes one or more hydroquinonesand/or one or more benzoquinones.
 16. The method of claim 5, wherein theone or more oxidants are electrochemically or electromagneticallygenerated in-situ.
 17. A composition comprising 1) a cellulosic orlignocellulosic material, 2) one or more compounds comprising one ormore naturally-occurring, non-radioactive group 5, 6, 7, 8, 9, 10 or 11elements, and, optionally, 3) one or more oxidants capable of increasingan oxidation state of at least some of said elements.
 18. A method ofreducing recalcitrance in cellulosic or lignocellulosic materials, themethod comprising: contacting a first lignocellulosic material having afirst level of recalcitrance with one or more ligninases and/or one ormore biomass-destroying organisms, to produce a second lignocellulosicmaterial having a second level of recalcitrance lower than the firstlevel of recalcitrance and contacting the second lignocellulosicmaterial with an enzyme and/or microorganism.
 19. The method of claim18, wherein the ligninases are selected from the group consisting ofmanganese peroxidases, lignin peroxidases, laccases and mixturesthereof.
 20. The method of claim 18, wherein the biomass-destroyingorganisms are selected from the group consisting of white rot, brownrot, soft rot and mixtures thereof.
 21. The method of claim 1, whereinthe method further comprises contacting the second cellulosic orlignocellulosic material with an enzyme and/or microorganism.
 22. Themethod of claim 1, wherein the method further comprises saccharifyingthe reduced recalcitrance material and then fermenting the saccharifiedmaterial.